Migration of mononuclear cells in the modified boyden chamber as evaluatedby DNA quantification and flow cytometry

In vitro migration of mononuclear cells in the modified Boyden chamber was evaluated using flow cytometry and DNA quantification (Hoechst 33258) of al l adherent and nonadherent cells. The effects of different membrane pore si zes, cell concentrations and incubation times were studied. Pore sizes of 3 and 5 mu m resulted in a reduction in the number of nonadherent cells comp ared with a pore size of 8 mu m. Reducing the incubation time from 60 to 40 and 20 min resulted in too few migrating monocytes for analysis by flow cy tometry. Flow cytometry showed that both monocytes and lymphocytes migrated and adhered to the membrane when using peripheral blood mononuclear cells (PBMC) to study monocyte migration. Migration of lymphocytes under these co nditions is a novel observation. A substantial number of migrated cells cou ld be identified by flow cytometry and quantified by DNA measurement as non adherent below the membrane. Samples of synovial fluid (n = 49) and plasma (n = 133) as chemoattractants analysed in triplicate resulted in mean coeff icient of variation (CV) values of 11 and 9%, respectively. Variation from assay to assay on the same day, using N-formyl-methionyl-leucyl-phenylanine (fMLP) 10(-7) m as chemoattractant resulted in a CV of 13%. Day-to-day var iation, using fMLP 10(-7) m as chemoattractant and the same well on three d ifferent days, resulted in a CV of 21%. These results were obtained using a pore size of 5 mu m, a PBMC concentration of 3 x 10(6)/ml and 60 min of in cubation. The combination of DNA quantification and flow cytometry thus all owed characterization and quantification of subsets of migrating adherent a s well as nonadherent mononuclear cells.